Nearly 10-15% of couples in our population who are of reproductive age are infertile. As a result, many infertile couples have opted for In vitro Fertilization (IVF), which was introduced in the United States (U.S.) in the early 1980s. According to the Centers for Disease Control and Prevention, over 140,000 cycles of IVF were performed in the U.S. alone in 2006 and this increased to almost 150,000 cycles in 2008 (cdc.gov/art). This suggests that the number of couples seeking IVF is on the rise and may persist if the general population continues to postpone having children. From these IVF cycles, it is estimated that more than a million embryos were produced annually, often with variable and poorly defined potential for successful implantation and development to term. Moreover, the average live birth rate per cycle following IVF was reported to be only 30% and this percentage has not significantly changed since the introduction of human IVF over 30 years ago (cdc.gov/art). Although the possible cause(s) of IVF failure are likely to be diverse, it is thought that chromosomal abnormalities, or aneuploidy, have contributed to nominal IVF success and live birth rates (Munne et al., (2003) Reprod Biomed Online, 7:91-97; Olgilvie (2008) Obstetrician & Gynaecol 10:88-92).
Previous studies have demonstrated that aneuploidy is present in 50-70% of cleavage-stage human embryos (Vanneste et al. (2009) Nat Med, 15:577-583; Johnson et al. (2010) Hum Reprod 25:1066-1075). While attempts have been made to correlate morphology with aneuploidy, it is well-known that aneuploid embryos often appear normal and suitable for transfer under traditional IVF assessment techniques (Baltaci et al. (2006) Reprod Biomed Online 12: 77-82). Currently, the most frequently used method for diagnosing aneuploidy is pre-implantation genetic screening (PGS) of day 3 biopsied blastomeres, which is invasive to the embryo, suffers from mosaicism and is utilized by only a small proportion of assisted reproduction patients (Kuo et al., (1998) J. Assist Reprod Genet, 15:276-280; Baart et al., (2006) Hum Repro 21:223-233). Alternative approaches such as extended culture of embryos to the blastocyst stage and analysis of chromosomal status via trophoectoderm biopsy have also been used to evaluate aneuploidy. However, additional potential risks including the introduction of epigenetic changes, embryo arrest and other factors that disrupt embryo integrity are thought to be associated with prolonged embryo culture (Khosla et al., (2001) Hum Reprod Update, 7: 419-427; Katari, et al. (2009) Hum Mol Genet, 18:3769-3778; Lim et al., (2009) Hum Reprod 24:741-747; Fernandez-Gonzalez et al., (2009) Reproduction 137:271-283.
The understanding in the art of basic embryo development is limited as studies on human embryo biology remain challenging and often exempt from research funding. Consequently, most of the current knowledge of embryo development derives from studies of model organisms. However, while embryos from different species go through similar developmental stages, the timing varies by species. These differences, and many others make it inappropriate to directly extrapolate from one species to another. (Taft, R. E. (2008) Theriogenology 69(1):10-16). The general pathways of human development, as well as the fundamental underlying molecular determinants, are unique to human embryo development. For example, in mice, embryonic transcription is activated approximately 12 hours post-fertilization, concurrent with the first cleavage division, whereas in humans the major wave of embryonic gene activation (EGA) occurs on day 3, around the 8-cell stage (Bell, C. E., et al. (2008) Mol. Hum. Reprod. 14:691-701; Braude, P., et al. (1988) Nature 332:459-461; Hamatani, T. et al. (2004) Proc. Natl. Acad. Sci. 101:10326-10331; Dobson, T. et al. (2004) Human Molecular Genetics 13(14):1461-1470). In addition, many of the genes that are modulated in early human development are unique (Dobson, T. et al. (2004) Human Molecular Genetics 13(14):1461-1470). Moreover, in other species such as the mouse, more than 85% of embryos cultured in vitro reach the blastocyst stage, one of the first major landmarks in mammalian development, whereas cultured human embryos have an average blastocyst formation rate of approximately 30-50%, with a high incidence of mosaicism and aberrant phenotypes, such as fragmentation and developmental arrest (Rienzi, L. et al. (2005) Reprod. Biomed. Online 10:669-681; Alikani, M., et al. (2005) Mol. Hum. Reprod. 11:335-344; Keltz, M. D., et al. (2006) Fertil. Steril. 86:321-324; French, D. B., et al. (2009) Fertil. Steril.). In spite of such differences, the majority of studies of preimplantation embryo development derive from model organisms and are difficult to relate to human embryo development (Zernicka-Goetz, M. (2002) Development 129:815-829; Wang, Q., et al. (2004) Dev Cell. 6:133-144; Bell, C. E., et al. (2008) Mol. Hum. Reprod. 14:691-701; Zernicka-Goetz, M. (2006) Curr. Opin. Genet. Dev. 16:406-412; Mtango, N. R., et al. (2008) Int. Rev. Cell. Mol. Biol. 268:223-290).
More recently, time-lapse imaging analysis has been implemented to monitor developing human embryos and potentially assess viability (Mio and Maeda (2008) Am J. Obstet Gynecol 199:660-665; Nakahara et al., (2010) J Assist Reprod Genet, 27:93-96). Besides providing a non-invasive approach to evaluate early embryo development and avoiding other limitations such as mosaicism, the detection and measurement of dynamic imaging parameters in human embryos may be accessible to all IVF patients. In these studies, developmental events including, fertilization, cleavage, blastocyst formation and embryo hatching, were analyzed and correlated with traditional IVF morphology criteria on day 3. However, no imaging parameters were correlated with blastocyst formation or pregnancy outcomes. Other methods have looked at the onset of first cleavage as an indicator to predict the viability of human embryos (Fenwick, et al. (2002) Human Reproduction, 17:407-412; Lundin, et al. (2001) Human Reproduction 16:2652-2657). However, these methods do not recognize the importance of the duration of cytokinesis or time intervals between early divisions.
Other methods have also used time-lapse imaging to measure the timing and extent of cell divisions during early embryo development (WO/2007/144001). However, these methods disclose only a basic and general method for time-lapse imaging of bovine embryos, which are substantially different from human embryos in terms of developmental potential, morphological behavior, molecular and epigenetic programs, and timing parameters surrounding transfer. For example, bovine embryos take substantially longer to implant compared to human embryos (30 days and 9 days, respectively). (Taft, (2008) Theriogenology 69(1):10-16. Moreover, no specific imaging parameters or time intervals are disclosed that might be predictive of human embryo viability.
More recently, time-lapse imaging has been used to observe human embryo development during the first 24 hours following fertilization (Lemmen et al. (2008) Reproductive BioMedicine Online 17(3):385-391). The synchrony of nuclei after the first division was found to correlate with pregnancy outcomes. However, this work concluded that the first cleavage was not an important predictive parameter, which contradicts previous studies (Fenwick, et al. (2002) Human Reproduction 17:407-412; Lundin, et al. (2001) Human Reproduction 16:2652-2657).
Finally, no studies have validated the imaging parameters through correlation with the molecular programs or chromosomal composition of the embryos. Methods of human embryo evaluation are thus lacking in several respects and can be improved by the present methods, which involve novel applications of time-lapse microscopy, image analysis, and correlation of the imaging parameters with molecular profiles and chromosomal composition. The inventors have surprisingly found that certain cell cycle imaging parameters not only predict embryo viability, but also aneuploidy including simple and complex mosaicism, monosomies and trisomies in human embryos through the correlation of imaging behavior with the chromosomal composition of the imaged embryos.